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. 2017 Jun 8;7:3057. doi: 10.1038/s41598-017-03130-z

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Indoxyl sulfate (IS), a uremic toxin, induces production of TNF-α by human monocytes. (A) Serum indoxyl sulfate (IS) and p-cresyl sulfate (PCS) levels in ESRD patients (n = 64) and healthy controls (n = 15) were quantified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). (B) Purified monocytes were stimulated with 1,000 μM of IS or 500 μM of PCS for 24 hr and then TNF-α mRNA expression was analyzed by real-time RT-PCR. (C and D) Purified monocytes were stimulated with IS at the indicated concentrations. The expression of TNF-α mRNA was analyzed by real-time RT-PCR after a 24 hr stimulation (C) and its protein level was quantified by ELISA at 48 hr post-stimulation (D). (E) Sera were pooled from patients with the top 10 (IS^higher-ESRD) and bottom 10 (IS^lower-ESRD) IS serum concentrations, respectively. As a control, sera from healthy controls were pooled. Monocytes isolated from healthy controls were treated with 30% (v/v) of the indicated sera for 24 hr and TNF-α mRNA expression was analyzed by real-time RT-PCR. Expression of β-actin was used as a normalization control. Bar graphs and scatter plot show the mean ± SEM of three (B), five to seven (C and D), and four (E) independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed unpaired (A) or paired t-test (B,C,D and E).